Microfluidic chip-based electrophoresis for DNA sequencing represents the future for high-throughput sequencing projects due to reductions in cost, time and reagent consumption and the possibility of integrating sequencing with other steps of genetic analysis into a total micro-analytical system. To this end, the development of optimal polymeric separation matrices and wall coatings for DNA sequencing on microfluidic chips is crucial.
Hydrophilic separation matrices, e.g., linear polyacrylamides (“LPA”), have been used along with covalent hydrophilic coatings to achieve read lengths greater than 500 bases by microchannel electrophoresis; however, the separations have all taken greater than 15-18 minutes. Poly(N,N-dimethylacrylamide) (“pDMA”) is a hydrophobic separation matrix that due to the hybrid separation mechanism achieves similar read lengths as polymers of the prior art but in much faster times. While covalent coatings have been used almost exclusively for all published microchannel DNA sequencing results, dynamic coatings, being much less expensive and also much easier to implement, are greatly preferred for the microchannel format. However, all dynamic coatings demonstrated for DNA sequencing have been somewhat hydrophobic leading to loss of separation efficiency due to interactions of the DNA fragments and the wall coatings. Previous work has shown that poly(N-hydroxyethylacrylamide) (“pHEA”) is a suitable hydrophilic dynamic coating for capillaries for both protein separation and DNA sequencing, but only when pHEA is also the separation matrix. While most published data on microchip-based DNA sequencing have reported read lengths of greater than 400 bases, sequencing times on chips generally have ranged from 18-30 minutes. And, capillary electrophoresis requires about 60-90 minutes to give comparable results. Time and read length considerations present ongoing concerns in the art relating to electrophoretic separations.